Recombinant Erythropoietin
Erythropoietin or EPO is a naturally occurring glycoprotein hormone produced by specialized epithelial cells lining the peritubular capillaries of the kidney nephrons. The protein itself consists of 193 amino acids within 4 discrete alpha helical polypeptides, the sequences of which are genetically encoded within the EPO gene on the long arm of chromosome 7, at cytogenetic location 77q22 (5). Though it has several other functions, erythropoietin's primary physiological purpose is to stimulate and increase the production of erythrocytes (red blood cells). Red blood cells, specifically their hemoglobin, are responsible for the blood's ability to transport oxygen and are absolutely essential to proper homeostasis. In those who are anemic due to chronic renal failure, inflammatory bowel disease (Crohn's disease and ulcer colitis), or myelodysplasia (abnormal myeloid stem cell growth/development) from chemotherapy or radiation, recombinant erythropoietin can be administered to increase red blood cell production and hopefully the patient's hematocrit (packed red cell volume in plasma) and ability to transport oxygen (1) (2) . It should be noted however that because erythropoietin increases one's hematocrit, it also increases one's blood viscosity which can lead to an increased risk of thrombogenesis (blood clot formation), embolism and all complications of embolism including stroke, myocardial infarction, gangrene, etc.(1). Erythropoeitin may also cause cardiovascular complications, including hypertension, and iron deficiency with prolonged used (2) . In the past few decades, recombinant erythropoietin has also been used illicitly as a substitute for autologous or homologous blood doping, though this practice is of course not endorsed by any of the scientists who help bring about the genesis of recombinant EPO (1). Recombinant erythropoietin (rHuEPO) was officially approved for medical use in the US by the FDA in 1992 and has since been regularly administered to those undergoing hemodialysis, though it is also occasionally prescribed in the other capacities described above (i.e. myelodysplasia) (7). Physiological Functions and Synthesis In Vivo Erythropoietin has a plethora of important physiological functions but its primary purpose is to stimulate erythrocyte differentiation and proliferation. Before we can truly appreciate the significance of erythropoietin, it is imperative to have a basic understanding of erythrocyte differentiation (view diagram at the right for a visual schematic). Erythrocytes, like all granulocyte leukocytes (basophils, eosinophils, and neutrophils), megakaryoctyes (which give rise to platelets) and monocytes, arise from the myeloid blood cell lineage. As in every cell differentiation process, the myeloid progenitor cells will differentiate into erythrocytes through differential gene expression and thus differential protein synthesis. One of the gene protein products unique to erythrocyte precursor cells is the erythropoietin receptor or Epo-R. If erythropoietin is not available to bind to this receptor, the erythrocyte precursor cell may undergo apoptosis but if the glycoprotein hormone is available, the cell will continue down the erythrocyte differentiation pathway to eventually become a mature red blood cell. Mature erythrocytes no longer express the erythropoietin receptor though other mature cell types, such as cells within the bone marrow and peripheral and central nervous systems, do. This coupled with autopsy findings may help to explain why erythropoietin affords the central nervous some protection from hypoxic (low blood oxygen) conditions. Erythropoietin is also involved in peripheral vasoconstriction and thus hypertension, angiogenesis and smooth muscle proliferation (1). In a healthy individual, serum erythropoietin levels will be insignificant but under hypoxic conditions (i.e. anemia), erythropoietin levels will increase 1,000-fold as the epithelial cells within the walls of the renal peritubular capillaries respond to the hypoxic insult. A small portion of this new circulating erythropoietin will have been manufactured by the liver, though the liver plays a much larger role in erythropoietin synthesis during fetal development. Erythropoietin gene expresion up-regulation is the result of a specific set of transcription factors termed hypoxia-inducible factors that are systematically degraded in the presence of oxygen (1). Recombinant Erythropoietin Synthesis and Purification Recombinant erythropoietin synthesis requires 2 procedures: #Gene extraction and vector synthesis #Protein Synthesis and purification Gene Extraction and Vector Synthesis While there are several ways one could hypothetically isolate and insert the EPO gene into a suitable vector, the procedure preformed by Fu-Kuen Lin et al. at the University of Chicago in 1985 will be discussed as a representative method. Researchers at the University of Chigcago first obtained a phage-borne human fetal liver genomic library (as you will recall, the liver is the primary generator of erythropoietin during fetal development) from an affiliated acedemic institution. Fu-Kuen and his team then introduced these phages to a bacterial culture and monitored the formation of phage plaques. As this was occuring, purified EPO from the urine of patients with aplastic anemia was subjected to protease digestion. The short peptide fragments generated through this digestion were then sequenced using an applied biosystems gas-phase microsequencer. The amino acid sequences of these fragments were then used to approximate the mRNA codon sequences responsible for erythropoietin in vivo synthesis. From these approximations, 2 oligonucleotide probe mixtures (EpV and EpQ) base on hexapeptide and heptapeptide sequences were synthesized using the phosphoramidite method. Each prepared probe mixture contained 128-oligonucleotide sequences to account for the degenerative nature of the genetic code. These synthetic probes were labeled using T4 polynucleotide kinases to catalyzes the transfer of a gamma-phostphate containing a radioactive P-32 isotope from a modified ATP nucleotide to the 5'-OH group of the oligonucleotide primers in question (4) (6). Once the phage plaques had been allowed adequate time to amplify, the phage particles were lysed so that their DNA could be subjected to further tests using the EPO oligonucleotide probes syntheisized previously. The DNA plasmids that hybridized with the radioactive probes were then inserted into the plasmid expression vector pDSVL. This plasma vector is unique in that it encodes the amino acid sequence of the protein dihydrofolate reductase (DHFR), an enzyme required for de novo synthesis of purine nucleotides, thymidine monophosphate and certain amino acids. The resulting pDSVL-gHuEPO plasmid was then introduced into a Chinese hamster ovarian cell line that had been genetically modified to omit the DHFR gene via calcium phosphate microprecipitate transfection. Cells that had obtained and were expressing this plasmid were then isolated using a medium lacking hypoxanthine (a purine derivative) and thymidine (a deoxyribose nucleoside joined to the pyrimidine base thymine). In such an environment, only cells expressing DHFR would be able to thrive. All of the mRNA within the cells expressing the pDSVL-gHuEPO plasmid was then isolated using oligo(dT)-cellulose and subjected to a Southern Blot assay. EPO cDNA probes that had been labeled with the radioactive isotope P-32 were then applied nylon membrane containing the mRNA separated during the previous gel electrophoresis step. The EPO coding mRNA was then processed using reverse transcriptase to produce a double stranded DNA erythropoietin gene devoid of introns. This gene was then inserted into Esherichia coli cultures using M13 phage vectors for further amplification. Protein Synthesis and Purification Recombinant erythropoietin can be synthesized in a variety of cell culture lines, including bacterial, mammalian (hamster) and even insect lines thanks to the recent isolation of the human erythropoietin gene. In each of these systems, the human erythropoietin gene is introduced into the cell culture from a previously prepared clonal library using species-specific viral vectors and translated into a protein using mammalian expression systems (i.e. mammalian ribosomes). The protein product itself is then isolated in a series of steps. The media containing the cell culture of interest is first concentrated and then subjected to a lytic buffer such as EDTA. The lysate from this previous step can then be run over a diethylaminoethyl cellulose (DEAE-C) resin derivative. Such resins are positively charged and can be used to isolate negatively charged biomolecules such as nucleic acids and proteins. Once the original supernate containing all hydrophobic cellular debris is remove and the product attached to the DEAE-C resuspended in an eluate solution, additional buffer is added before the entire solution is concentrated. The concentrate then undergoes high-performance liquid chromatography (HPLC). As the procedure proceeds, small fractions of the eluate solution are collected within vials containing small amounts of glucoside and dried using vacuum suction. HPLC eluate fractions shown to contain erythropoietin are identified using various molecular means (including Western Blot and bioassays) will again be dissolved in an ionic solution before being applied to a concanavalin A-agarose column. The column is then washed with a buffer solution and the eluate collected is said to contain recombinant erythropoietin protein (3). References 1. Wikipedia Erythropoietin Article 2. What is human recombinant erythropoietin? 3. High-level expression and purification of a recombinant human erythropoietin produced using a baculovirus vector 4. Cloning and expression of the Human Erythropoietin Gene 5. Genetics Home Reference 6. T4 Polynucleotide Kinase 7. Access to recombinant erythropoietin by Medicare-entitled dialysis patients in the first year after FDA approval